Project Summary/Abstract The long-term goal of our program is to develop radiotracers that can be used for non-invasive PET imaging to detect and localize bacterial pathogens in humans. Such radiotracers will distinguish between different pathogen populations, serve as non-invasive diagnostics and inform on bacterial load during chemotherapy, thereby identifying and improving treatment of patients with infectious diseases. While this approach will ultimately be applicable to any infectious agent, the current proposal is focused on the development of imaging agents for Staphylococcus aureus, which is the leading cause of skin and soft tissue infections, osteomyelitis, infective endocarditis, bacteremia and device-related infections. Osteomyelitis and endocarditis, which are infections of bones and heart valves, respectively, as well as prosthetic joint infections, are particularly difficult to detect and diagnose due to their location in the human body which limits the availability of clinical samples, and are a major cause of morbidity, mortality and financial costs. Surgical intervention is often required, and the inappropriate use of broad spectrum antibiotics leads to additional problems such as disruption of the microbiome. Although non-invasive imaging is a promising approach for detecting and diagnosing infection, current clinical methods lack sensitivity and specificity, in many cases being unable to distinguish infection from inflammation. In addition, current PET infection tracers under development either are not taken up by S. aureus, suffer from poor signal-to-background ratios, or have not been extensively evaluated in S. aureus infection models. In this proposal, we describe the evaluation of fluorine-18 labeled 2-fluoro-4-aminobenzoic acid ([18F]F-PABA), a novel radiotracer that is selectively taken up by bacteria. We have shown that [18F]F-PABA accumulates at the site of S. aureus soft tissue infection in an animal model of disease and can distinguish bacterial infection from inflammation. In Aim 1 we will determine the ability of [18F]F-PABA to image clinically relevant strains of S. aureus and assess the ability of [18F]F-PABA to monitor the progression of infection and to quantify bacterial burden during antibiotic therapy. In Aim 2 we will determine the ability of [18F]F-PABA to detect and image S. aureus in two clinically-relevant models of infection, osteomyelitis and infective endocarditis. In Aim 3 we will determine which bacterial species can take up [18F]F-PABA including those that are resistant to drugs that target folate biosynthesis, determine the fate of F-PABA in bacteria cells in order to understand the molecular factors that control F-PABA accumulation in bacteria, and identify structural modifications that improve the ability of [18F]F-PABA to image infection.